Exported proteins in bacteria participate in many diverse and essential cell functions such as motility, signal transduction, macromolecular transport and assembly, and the acquisition of essential nutrients. For pathogenic bacteria, many exported proteins are virulence determinants that function as adhesins to colonize and thus infect the host or as toxins to protect the bacteria against the host's immune system (for a review, see Hoepelman and Tuomanen, 1992, Infect. Immun. 60:1729-33).
Since the development of the smallpox vaccine by Jenner in the 18th century, vaccination has been an important armament in the arsenal against infectious microorganisms. Prior to the introduction of antibiotics, vaccination was the major hope for protecting populations against viral or bacterial infection. With the advent of antibiotics in the early 20th century, vaccination against bacterial infections became much less important. However, the recent insurgence of antibiotic-resistant strains of infectious bacteria has resulted in the reestablishment of the importance of anti-bacterial vaccines.
One possibility for an anti-bacterial vaccine is the use of killed or attenuated bacteria. However, there are several disadvantages of whole bacterial vaccines, including the possibility of a reversion of killed or attenuated bacteria to virulence due to incomplete killing or attenuation and the inclusion of toxic components as contaminants.
Another vaccine alternative is to immunize with the bacterial carbohydrate capsule. Presently, vaccines against Streptococcus pneumoniae employ conjugates composed of the capsules of the 23 most common serotypes of this bacterium. these vaccines are ineffective in individuals most susceptible to pathological infection--the young, the old, and the immune compromised--because of its inability to elicit a T cell immune response. A recent study has shown that this vaccine is only 50% protective for these individuals (Shapiro et al., 1991, N. Engl. J. Med. 325:1453-60).
An alternative to whole bacterial vaccines are acellular vaccines or subunit vaccines in which the antigen includes a bacterial surface protein. These vaccines could potentially overcome the deficiencies of whole bacterial or capsule-based vaccines. Moreover, given the importance of exported proteins to bacterial virulence, these proteins are an important target for therapeutic intervention. Of particular importance are proteins that represent a common antigen of all strains of a particular species of bacteria for use in a vaccine that would protect against all strains of the bacteria. However, to date only a small number of exported proteins of Gram positive bacteria have been identified, and none of these represent a common antigen for a particular species of bacteria.
A strategy for the genetic analysis of exported proteins in E. coli was suggested following the description of translational fusions to a truncated gene for alkaline phosphatase (phoA) that lacked a functional signal sequence (Hoffman and Wright, 1985, Proc. Natl. Acad. Sci. U.S.A. 82:5107-5111). In this study, enzyme activity was readily detected in strains that had gene fusions between the coding regions of heterologous signal sequences and phoA indicating that translocation across the cytoplasmic membrane was required for enzyme activity. Subsequently, a modified transposon, TnphoA, was constructed to facilitate the rapid screening for translational gene fusions (Manoil and Beckwith, 1985, Proc. Natl. Acad. Sci. U.S.A. 82:8129-8133). This powerful tool has been modified and used in many Gram negative pathogens such as Escherichia coli (Guitierrez et al., 1987, J. Mol. Biol. 195:289-297), Vibrio cholera (Taylor et al., 1989, J. Bacteriol. 171:1870-1878), Bordetella pertussis (Finn et al., 1991, Infect Immun. 59:3273-9; Knapp and Mekalanos, 1988, J. Bacteriol. 170:5059-5066) and Legionella pneumophila (Albano et al., 1992, Mol. Microbiol. 6:1829-39), to yield a wealth of information from the identification and characterization of exported proteins. A similar strategy based on gene fusions to a truncated form of the gene for .beta.-lactamase has been used to the same end (Broome-Smith et al., 1990, Mol. Microbiol. 4:1637-1644). A direct strategy for mapping the topology of exported proteins has also been developed based on "sandwich" gene fusions to phoA (Ehrmann et al., 1990, 87:7574-7578).
For a variety of reasons, the use of gene fusions as a genetic screen for exported proteins in Gram positive organisms has met with limited success. Plasmid vectors that will create two or three part translational fusions to genes for alkaline phosphatase, .beta.-lactamase and a-amylase have been designed for Bacillus subtilis and Lactococcus lacti (Payne and Jackson, 1991, J. Bacteriol. 173:2278-82; Perez et al., 1992, Mol. Gen. Genet. 234:401-11; Smith et al., 1987, J. Bacteriol. 169:3321-3328; Smith et al., 1988, Gene 70:351-361). Gene fusions between phoA and the gene for protein A (spa) from Staphylococcus aureus have been used to determine the cellular localization of this protein (Schneewind et al., 1992, Cell. 70:267-81). In that study, however, enzyme activity for alkaline phosphatase was not reported.
Mutagenesis strategies in several streptococcal species have also been limited for several reasons. Efficient transposons similar to those that are the major tools to study Gram negative bacteria have not been developed for streptococcus. Insertion duplication mutagenesis with non-replicating plasmid vectors has been a successful alternative for Streptococcus pneumoniae (Chen and Morrison, 1988, Gene. 64:155-164; Morrison et al., 1984, J. Bacteriol. 159:870). This strategy has led to the mutagenesis, isolation and cloning of several pneumococcal genes (Alloing et al., 1989, Gene. 76:363-8; Berry et al., 1992, Microb. Pathog. 12:87-93; Hui and Morrison, 1991, J. Bacteriol. 173:372-81; Lacks and Greenberg, 1991, Gene. 104:11-7; Laible et al., 1989, Mol. Microbiol. 3:1337-48; Martin et al., 1992, J. Bacteriol. 174:4517-23; McDaniel et al., 1987, J. Exp. Med. 165:381-94; Prudhomme et al., 1989, J. Bacteriol. 171:5332-8; Prudhomme et al., 1991, J. Bacteriol. 173:7196-203; Puyet et al., 1989, J. Bacteriol. 171:2278-2286; Puyet et al., 1990, J. Mol. Biol. 213:727-38; Radnis et al., 1990, J. Bacteriol. 172:3669-74; Sicard et al., 1992, J. Bacteriol. 174:2412-5; Stassi et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:7028-7032; Tomasz et al., 1988, J. Bacteriol. 170:5931-5934; Yother et al., 1992, J. Bacteriol. 174:610-8).
Of note in the search for exported pneumococcal proteins that might be attractive targets for a vaccine is pneumococcal surface protein A (PspA) (see Yother et al., 1992, supra). PspA has been reported to be a candidate for a S. pneumoniae vaccine as it has been found in all pneumococci to date; the purified protein can be used to elicit protective immunity in mice; and antibodies against the protein confer passive immunity in mice (Talkington et al., 1992, Microb. Pathog. 13:343-355). However, PspA demonstrates antigenic variability between strains in the N-terminal half of the protein, which contains the immunogenic and protection eliciting epitopes (Yother et al., 1992, supra). This protein does not represent a common antigen for all strains of S. pneumoniae, and therefore is not an optimal vaccine candidate.
Recently, apparent fusion proteins containing PhoA were exported in species of Gram positive and Gram negative bacteria (Pearce and Masure, 1992, Abstr. Gen. Meet. Am. Soc. Microbiol. 92:127, abstract D-188). This abstract reports insertion of pneumococcal DNA upstream from the E. coli phoA gene lacking its signal sequence and promoter in a shuttle vector capable of expression in both E. coli and S. pneumoniae, and suggests that similar pathways for the translocation of exported proteins across the plasma membranes must be found for both species of bacteria.
Recent studies have shown that genetic transfer in several bacterial species relies on a signal response mechanism between individual cells. Conjugal plasmid transfer is mediated by homoserine lactones in Agrobacterium tumifaciens (Zhang et al., 1993, Scinece 362:446-448) and by small secreted polypeptides in Enterococcus faecalis (for a review, see Clewell, 1993, Cell 73:9-12). Low molecular weight peptide activators have been described which induce transformation in S. pneumoniae (Tomasz, 1965, Nature 208:155-159; Tomasz, 1966, J. Bacteriol. 91:1050-61; Tomasz and Mosser, 1966, Proc. Natl. Acad. Sci. USA 55:58-66) and Streptococcus sanguis (Leonard and Cole, 1972, J. Bacteriol. 110:273-280; Pakula et al., 1962, Acta Microbiol. Pol. 11:205-222; Pakula and Walczak, 1963, J. Gen. Microbiol. 31:125-133). A peptide activator which regulates both sporulation and transformation has been described for B. subtilis (Grossman and Losick, 1988, Proc. Natl. Acad. Sci. USA 85:4369-73). Furthermore, genetic evidence suggests that peptide permeases may be mediating these processes in both E. faecalis (Ruhfel et al., 1993, J. Bacteriol. 175:5253-59; Tanimoto et al., 1993, J. Bacteriol. 175:5260-64) and B. subtilis (Rudner et al., 1991, J. Bacteriol. 173:1388-98).
In S. pneumoniae, transformation occurs as a programmed event during a physiologically defined "competent" state. Induced by an unknown signal in a density dependent manner, cells exhibit a single wave of competence between 5.times.10.sup.6 and 1-2.times.10.sup.7 cfu/ml which is the beginning of logarithmic growth (Tomasz, 1966, supra). With induction, a unique set of competence associated proteins are expressed (Morrison and Baker, 1979, Nature 282:215-217) suggesting global regulation of transformation associated genes. Competent bacteria bind and transport exogenous DNA, which if homologous is incorporated by recombination into the genome of the recipient cell. Within one to two cell divisions, the bacteria are no longer competent. As with induction, inactivation of competence occurs by an unknown mechanism.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.